KR100458978B1 - The blood glucose strip with low sensitivity to hematocrit - Google Patents

Abstract

A reagent strip for measuring glucose concentration in whole blood lowers the interference of hematocrit during glucose measurement. When the biological fluid comes in contact with the script, it causes a color change, which is a measure of the glucose concentration in the fluid, among the reagents impregnated in the strip. However, color change is also affected by the red cell concentration (hematocrit), which reduces the accuracy of glucose measurements. The hematocrit effect is reduced by adding an acrylic acid polymer to the reagent.

Description

The blood glucose strip with low sensitivity to hematocrit

The present invention relates to a dry phase reagent strip for measuring glucose concentration in a whole blood sample, more particularly to a relatively less sensitive strip for the hematocrit of a blood sample.

Dry phase reagent strips incorporating enzyme-based compositions are widely used in clinical laboratories, clinics, hospitals, and homes to determine the glucose concentration in test samples of biological fluids. Indeed, reagent strips are becoming a daily necessity in the multitude of millions of people with diabetes worldwide. Because diabetes can cause dangerous abnormalities in blood chemistry, it can lead to blindness, renal failure, and other serious medical consequences. In order to minimize the risk of this consequence, most diabetics should adjust their glucose levels accordingly, for example by dietary control and / or insulin injections, after periodically measuring their glucose levels themselves. Some patients should measure their blood glucose levels more frequently than four times a day.

Rapid, inexpensive and accurate reagent strips for glucose measurement are crucial for diabetics who need to regulate their own diets to regulate sugar intake and / or inject insulin and have to measure blood glucose levels frequently in this regard.

The reagent strip is known to contain indicators that vary in color depending on the concentration of glucose in the blood sample applied to the strip. Some of these strips include enzymes such as glucose oxidase that can oxidize glucose to gluconate lactone and hydrogen peroxide. They also contain oxidizable dyes and substances with peroxidation activity that can selectively catalyze the oxidation of oxidizable dyes in the presence of hydrogen peroxide (see Phillips et al., Issued June 19, 1990 U.S. Patent No. 4,935,346].

Regardless of whether the measurement is performed at home, clinic, clinic or hospital, the accuracy and reproducibility of glucose measurements is very important. In the case of a color-indicating reagent strip, it is preferable that the color change is clear and not sensitive to various compounds contained in blood in addition to glucose. In the case of reagent strips that are read by the naked eye, it is especially important for diabetics who suffer from visual impairment to have a test reagent that exhibits significant color change depending on the glucose concentration. The color change caused by changes in absorption and / or reflection at the wavelengths is also important in the accuracy of the metering-reading strip.

The performance of reagent strips can be influenced by the presence of various interfering factors and the need to reduce or eliminate interference effects in clinical chemistry has been recognized by other literature. For example, in European Patent Application No. 93111290.8, published Jan. 19, 1994, Arter et al. Describe analytical elements for the determination of analytes in aqueous fluids. This member comprises a reagent capable of binding a glass sulfhydryl group present in the fluid. Arter et al. Are mainly interested in detecting analytes such as acetaminophen, salicylate, creatinine, cholesterol, HDL cholesterol, triglyceride, glucose and uric acid. They use enzymes that generate hydrogen peroxide, which is verified by peroxidase-coupled redox chemistry. Interfering sulfhydryl groups, such as N-acetylcysteine, present in the serum of a particular type of drug treatment individual may be present in the fluid, which may cause oxidation / reduction even in the absence of analytes. Arthur et al. Have attempted to solve the problem of the interfering glass sulfhydryl group by adding an agent that reacts with the sulfhydryl group. Suitable formulations include maleimide, N-ethylmaleimide, iodoacetamide, silver nitrate and chloride.

U.S. Patent No. 5,185,247 to Ismail et al., Issued February 9, 1993, describes an enzyme-based test strip that is stabilized by the addition of various compositions. These include imidazoles in formulations which confer thermal stability, but this is only effective if the strip is also impregnated with an "ascorbate intercalating composition" which also contains mercuric oxide and sarcosine. In general, incorporation of heavy metal oxides in reagents is undesirable because they are toxic.

European Patent Application No. 93303643.6, published on Dec. 15, 1993 by Matzinger et al., Describes a reagent strip for measuring the concentration of glucose in whole blood. The strip has a testing pad that includes a reagent system that displays the glucose concentration with a color change. The test pad is formed from an anisotropic film in which a relatively large pore is present near one major surface and a smaller pore is present near the opposite surface. The porous carrier medium is attached to the large voided surface of the pad. A whole blood sample is applied to a transport medium that carries a detectable sample volume on the large pore side of the pad. Glucose in the sample then moves toward the opposite side, where glucose reacts with the reagent and is visible from the surface with the small pore of the pad, causing a color change indicative of the glucose concentration in the sample.

U.S. Patent No. 5,306,623 to Kiser et al. Describes a blood glucose test strip comprising a separation matrix for separating red blood cells from whole blood. The test reagent reacts with the separated components and causes a color change indicating the blood glucose concentration. Of the components incorporated in the separating layer, a polyacrylic polymer (Carbopol ^R ) is described. These "separate components" (also referred to as "hematocrit modulators" without further description) are generally present in separate separation layers, while Kaiser et al. Also include reagents containing both signal generating components and blood separating components Single layer matrix. In all cases, the separation component is included in an amount ranging from 7 to 35% w / v. Indeed, carbopole can not be used in this concentration range in this reagent.

It is known that the interference factor can affect the color change of the reagent strip measuring blood glucose. For example, degraded color changes in glucose measurements are generally associated with the concentration of hemocytes in the blood (hematocrit).

When applied to blood containing glucose, there has been a continuing need for reagent strips whose color changes obviously depending on the glucose concentration. Ideally, the reagent strip should accurately measure the glucose concentration in the range of about 40 to 500 mg / dL even when applied to whole blood samples with a hematocrit concentration in the range of 25 to 60%.

According to the present invention, a reagent strip for measuring the glucose concentration in a whole blood sample containing red blood cells

(a) an anisotropic film having a sample surface having a relatively large gap and a test surface having a relatively small gap and having a sample accommodated on the sample surface and adjusted to pass toward the test surface, and

(b) (i) a component that produces hydrogen peroxide from glucose and oxygen,

    (ii) changes in hue by reaction with hydrogen peroxide, and

    (iii) an acrylic acid polymer at a concentration of about 0.1% to about 4% w / v, which reduces the effect of red blood cells on the glucose concentration measurement.

In the method of the present invention, a method of measuring the glucose concentration in a whole blood sample containing red blood cells comprises the steps of (a) (i) having a sample surface having a relatively large pore and a test surface having a relatively small pore size, (ii) To change the hue of the test surface and can be impregnated with a test reagent comprising a glucose oxidase, a peroxidase, an oxidizable dye or dye couple, and an acrylic acid polymer having a concentration range of from about 0.1% to about 4% w / v Providing an anisotropic film;

(b) applying blood to the sample side of the membrane and

(c) measuring the blood glucose concentration by measuring the color change of the test surface.

The strip is adjusted to accommodate whole blood samples, including red blood cells and glucose, applied on the sample surface. It is not necessary to measure the sample volume or adjust it within the appropriate limits. The porosity of the membrane allows the fluid to pass from the sample side to the test side, for example, by capillary action. That is, the test reagent may react with blood glucose to cause a color change on or near the test surface. The color change depends on the concentration of glucose in the sample. The blood glucose concentration can be measured by various methods, for example, by comparing the color of the test surface with a calibration reference or color chart, by using a reflective metering method, or by using a series of segments corresponding to the glucose concentration range, Or by measuring the glucose concentration by recognizing the final segment that changes color within the cell. Dark-colored red blood cells, which make detection of color change difficult, are filtered near the sample surface of the anisotropic film which is graded and arranged from large pores on the sample surface to small pores on the test surface. Various materials can be used for various components of the reagent strip of the present invention. Some of these materials are described in U.S. Patent Nos. 5,306,623 and 5,418,142, issued April 26, 1994 and May 23, 1995, respectively, to Kaiser et al., Which is incorporated herein by reference.

The test reagent comprises one component such as glucose oxidase for converting glucose to hydrogen peroxide and one or more components for detecting hydrogen peroxide produced from glucose present in the sample. The component for detecting hydrogen peroxide may be a peroxidase, preferably a horseradish peroxidase combined with an "indicator" that changes color during the course of the reaction. The indicator may be an oxidizable dye or a dye couple. The peroxidase acts as a catalyst in the oxidation reaction of the indicator in the presence of hydrogen peroxide.

The test reagent further comprises an acrylic acid polymer that reduces the effect of varying the glucose concentration at which the hematocrit (the percentage of red blood cells in the whole blood sample) is measured.

The test strips and methods of the present invention allow glucose concentrations to be measured in a blood sample having a glucose concentration in the range of about 40 to 500 mg / dL and a hematocrit level in the range of about 25 to 60%.

The present invention relates to a dry phase reagent test strip for measuring the concentration of blood glucose in whole blood. The main component of such a strip is a porous membrane incorporating a test reagent. This strip changes color in response to glucose in the blood sample applied to the strip.

The membrane may be a homogeneous composition or a coated substrate. It has a sample surface on which the sample is applied and a test surface on which the color change is observed. Preferably, the membrane is anisotropic and more preferably has a wide pore size. For example, the ratio of maximum void to minimum void has a pore size gradient in the range of about 100 to 400. On one side of the membrane where the void is the minimum size, the void volume is relatively small and the material of the membrane is generally very dense in the layer, which typically can constitute less than 20% of the film thickness. Within this layer, the pore size is preferably in the range of about 0.1 to about 0.8 microns, and the nominal pore size is preferably about 0.3 to 0.4 microns. In terms of having large pores, the pore size is preferably in the range of about 40 to about 125 占 퐉. When the blood is applied to the sample (with large pores), the sample will hit the pores that gradually become smaller as they penetrate the membrane. Ultimately, solids such as red blood cells reach locations within the membrane that are no longer permeable. The remainder of the sample, still containing dissolved glucose, passes through to the test surface. The anisotropy of the membrane is to allow the membrane to pass at a relatively rapid flow rate even if filtration of solids occurs.

As the sample passes through the membrane, the reaction with the reagent causes the light absorbing dye to form or decolorize within the volume of space near the test surface, substantially affecting the reflection from the film.

Polysulfone and polyamide (nylon) are examples of suitable membrane materials. Other polymers having comparable properties can also be used. Since the polymer can be modified to introduce other functional groups that provide a charged structure, the surface of the membrane may be neutral, positive or negative.

A preferred method for preparing the porous material forming the membrane is to cast the polymer without a support core. Such a membrane is, for example, an anisotropic polysulfone membrane commercially available from Memtec Inc., Timonium, MD. A film having a thickness of less than about 200 탆 is generally used, but a film having a thickness of about 115 to 155 탆 is preferable. In particular, when the membrane is nylon or anisotropic polysulfone, it is most preferable that the thickness is about 130 to 140 mu m.

The membrane is saturated by immersing the membrane in a mixture of components to treat the membrane with the test reagent. Preferably, at least a portion of the components are applied successively to the film. Excess reagents can be removed by mechanical means such as, for example, air knife, doctor blade or glass rod. The membrane is then dried. Reagents tend to be concentrated near the (test) side of the membrane with small pores.

The test reagents include (i) a component that converts glucose to hydrogen peroxide, (ii) a component that detects hydrogen peroxide, and (iii) a component that reduces the effect of the hematocrit in the blood sample on glucose concentration measurements. The reagent may also optionally include a separation component that efficiently removes solids from the biological fluid by allowing solids such as red blood cells to adhere to or capture within the membrane. Additional components may also be included as described below and in the Examples.

Preferred components for converting glucose to hydrogen peroxide include glucose oxidase, an enzyme generally obtained from Aspergillus niger or Penicillium. Glucose oxidase reacts with glucose and oxygen to produce gluconolactone and hydrogen peroxide. The optimum concentration of glucose oxidase depends on the composition of the indicator system. For example, when the indicator system is MBTHSB-ANS (described below), a glucose oxidase in the range of about 500 to 10,000 U./mL is suitable, more preferably about 700 to 2000 U./mL, more preferably about 1000 U./mL Is most preferable. In general, high concentrations of glucose oxidase lead to a more rapid reversal of the reaction.

The hydrogen peroxide thus produced reacts with a component for the detection of hydrogen peroxide, including peroxidase, which selectively catalyzes the reaction between hydrogen peroxide and indicator. Peroxidase uses hydrogen peroxide as an oxidizing agent capable of removing hydrogen atoms from various substrates. Suitable peroxidases may include perit protoporphyrins, which are the red hemin obtained from plants. Peroxidases obtained from animals, for example from the thyroid of animals, are also suitable. As a component of the component for detecting hydrogen peroxide, horseradish peroxidase is particularly preferred.

Preferably the hydrogen peroxide catalysed by the peroxidase reacts directly or indirectly to form or decompose the indicator dye which absorbs the light within a predetermined wavelength range. Preferably, the indicator dye is strongly absorbed at a wavelength different from the wavelength at which the sample or test reagent strongly absorbs. The oxidized form of the indicator may be a colored, pale or colorless end product, which demonstrates the color change of the test side of the membrane. That is, the test reagent may indicate the presence of glucose in the sample as the staining area is bleached or the colorless area is stained.

Indicators useful in the present invention include both a one-component and two-component chromogenic substrate. One-component systems include aromatic amines, aromatic alcohols, azines, and benzidine, such as tetramethylbenzidine-HCl. Suitable two-component systems include those in which one component comprises MBTH, an MBTH derivative (e.g., as described in U.S. Patent Application Serial No. 08 / 302,575, incorporated herein by reference) or 4-aminoantipyrine and the other component comprises an aromatic amine, an aromatic An alcohol, a conjugated amine, a conjugated alcohol or an aromatic or aliphatic aldehyde. Examples of two-component systems include 3-methyl-2-benzothiazolinone hydrazone hydrochloride (MBTH) combined with 3-dimethylaminobenzoic acid (DMAB); (3-methyl-2-benzo-thiazolinone) compounded with MBTH and 8-anilino-1-naphthalene sulfonic acid ammonium (ANS) combined with 3,5-dichloro-2-hydroxybenzenesulfonic acid Hydrazone] N-sulfonylbenzenesulfonate monosodium (MBTHSB). MBTHSB-ANS is preferred.

The component for reducing the effect of hematocrit in the blood sample during glucose measurement is acrylic acid polymer (-CH2-CH-COOH) _n . Such polymers are generally known under the trade name CARBOPOL. Preferably, the molecular weight of the polymer is from about 200,000 to about 4,000,000. A molecular weight of about 750,000 is most preferred. A preferred concentration is in the range of about 0.1 to 4.0% w / v and most preferably about 0.5% w / v.

It is particularly preferred that the method of the present invention is carried out using a biological fluid having a glucose concentration in the range of about 50 to 400 mg / dL. The method of the present invention demonstrates a greater degree of color change per unit of glucose concentration in a sample when used within the preferred glucose concentration range, especially when the hematocrit concentration is within the preferred range of 25-60% . The advantageous effect is greater as the glucose concentration is higher.

In a preferred embodiment of the reagent strip of the present invention, the reagent comprises an inhibitor to inhibit the color-changing reaction. In this embodiment, the test reagent coated on the membrane or immersed in the membrane is not uniform on the surface of the test strip. Moreover, the reagent is preferably applied to the membrane in a series of parallel stripes or applied to the "result segment" (where the concentration of the inhibitor in the composition in the adjacent resultant segment increases stepwise). Thus, each successive segment should be able to change the color of the segment step by step as the glucose concentration in the sample increases. The strip of this design is "direct reading "; In other words, this enables visual and direct quantitative readings. Details of this aspect are found in co-pending U.S. Patent 411,238, filed March 27, 1995, herein incorporated by reference.

In another aspect of the invention, the reagent does not contain an inhibitor, but the strip includes a porous transport medium of the second layer attached to the sample side of the membrane, in addition to the membrane impregnated with the reagent. In this "bi-layer" embodiment, the transport medium is adapted to receive a whole blood sample and carry the detectable site of the sample to the sample receiving surface of the membrane. This sample can be moved by capillary action. The transport medium preferably extends past one or more ends of the test pad to form a reservoir that receives an excess of blood sample. Thus, it is preferred that the carrier medium contains about 10 to about 50 microliters of blood, preferably about 35 microliters of blood, and about 3 to about 10 microliters of blood through the membrane. The carrier medium may be composed of natural fibers (e.g. cotton or paper), and polyester, polyamide, polyethylene and other synthetic polymers. Polyethylene, e. G. Porous polyethylene, available from Porex Corporation (Porex Corp., Fairburn, Georgia) is the preferred transport medium material. Optionally, this aspect of the reagent strip may include a support layer with an apeture capable of measuring the color change attached to the test surface of the membrane. The color change on the test surface of the film is preferably measured with an optical sensor to obtain the blood glucose concentration.

When measured by an optical sensor (i.e., a reflection metering method), one or more calibration reference color indicators are exposed to the light source either before or after the test surface is exposed to the light source. For example, light may be continuously reflected from the color guide and the test surface to the sensor.

The sensor generates a signal that changes as the reference and test surface are exposed to the light source in succession. The change in signal is then quantitatively related to the concentration of glucose in the sample according to a previously prepared formula using similar observation means and samples for which the glucose concentration is known. Details of such embodiments are found in U.S. Patent No. 493,435, filed June 22, 1995, and incorporated herein by reference.

The present invention will now be described in more detail with reference to the drawings. Figure 1 shows a membrane (10) of the present invention for measuring the amount of glucose in a whole blood sample. Although shown in a curved form, the membrane 10 is flexible and generally flat in use. The membrane includes a sample surface 12 to which a blood sample is applied and a test surface 14 that exhibits the presence of glucose with a change in hue on its own or in its surroundings. The color change results from the interaction of the glucose with the reagent impregnated in the void 16. Preferably, the pore size is relatively large near the sample surface 12 and decreases in size as it approaches the test surface 14. Such a pore size gradient causes the red blood cells to be captured near the sample surface 12 so that their color has as little effect as possible on their ability to observe the color change indicative of the presence of glucose.

For a direct reading test strip, the membrane of Figure 1 is sandwiched between two cover sheets, which may be any suitable thermoplastic film well known in the art. 2 and 3 are plan views of the sample surface 22 and the test surface 24 of the test strip 20, respectively. When in use, the blood sample is applied to the opening 26 of the sample surface 22. The sample is vertically diffused into the top and bottom of the strip by capillary action and penetrates the membrane. The vent hole 28 facilitates sample flow into the strip. It is confirmed by observing the sample through any transparent windows 30 and 32 whether a sufficient amount of sample is provided to measure. On the test surface 24, an indicator ring, such as (34), is labeled to indicate the glucose concentration in the blood entering the oxygen required for the color forming reaction. As the test progresses, when the blood glucose concentration in the sample satisfies or exceeds the amount corresponding to the ring, the indicator changes color continuously. Any chemical timer circle 36 indicates that the proper time has elapsed to read the strip (details of the timer are disclosed in co-pending U.S. Patent 411,238, filed March 27, 1995 Lt; / RTI > The results shown in Figure 3 indicate that the sample glucose concentration is above 120 mg / dL but below 150 mg / dL. It is noted in Fig. 3 that the hue change indicates that it is colored from white by reaction with glucose. However, the system can also work with indicator dyes that are degraded by oxidation induced by glucose to change the corresponding color change from color to white.

4 is a schematic view of a bi-layered reagent strip with a large voided surface of the membrane 10 of FIG. 1 attached to a transport medium 40. FIG. The surface with the small pores is attached to any support 42. The support 42 has a sphere 44 allowing an optical sensor (not shown) to detect the color developed by the reaction of the reagent of the membrane 10 with the glucose in the blood sample on the test surface 46.

The following examples demonstrate the efficacy of an acrylic acid polymer to reduce the sensitivity of a reagent strip to changes in hematocrit concentration in a sample according to the present invention. The present embodiment is not limited in any way.

Example

Some grades of polyacrylic acid are available from Aldrich Chemical (Milwaukee, Wis.) And used without purification. Carbopol ^R 910 (polyacrylic acid) obtained from BF Goodrich, Cleveland, OH is used as a reference material. The polyacrylic acids obtained from Aldrich and Viehfelder Leich indicate very similar physical and chemical properties.

The polyacrylic acid is suspended in acetonitrile and slowly dispersed in the stirred enzyme solution (see Table 1). The resulting mixture is allowed to react for 1 hour, after which the solution is adjusted to 0.1M disodium citrate. The resulting yellowish brown solution is coated onto a polysulfone membrane manufactured by Memtec Corp. (San Diego, Calif.). When the coated membrane is dried in an air circulating oven at 56 ° C for 10 minutes, its color changes to light yellow during this time. The dried film is coated with a dye solution (Table 2) and dried again as described above. The membrane is then cut into strips for blood testing.

[Table 1]

Enzyme solution

[Table 2]

Dye solution

Three strips of polyacrylic acid with different molecular weights and Carbopol 910 were prepared and used to measure the glucose concentration in whole blood at three hematocrit values of 25%, 43% and 60%. The results are shown in Table 3 together with the results obtained using a control without polyacrylic acid. Polyacrylic acid generally has a degree of polymerization inhibition depending on the degree of polymerization. As the molecular weight increases, the effect of the hematocrit generally decreases but the time it takes for the reaction to complete increases and the color intensity decreases. Also, high molecular weight polymers are less soluble, and such properties are undesirable in the manufacturing process. That is, the optimum molecular weight of the polyacrylic acid is selected from the intermediate value of about 750,000.

[Table 3]

Hematocrit Performance of Glucose Test Strips Containing Polyacrylic Acid

Those skilled in the art will appreciate that the techniques and embodiments described above are listed for carrying out the invention and are not intended to limit the invention. Changes in the details set forth herein may be made without departing from the spirit and scope of the invention.

The reagent strips of the present invention enable comparatively simple and rapid measurement of glucose concentration in blood samples not measured over the concentration range of glucose. This reagent strip provides a substantially constant glucose concentration in a whole blood sample in which the hematocrit ranges from about 25% to 60% concentration.

Figure 1 is a perspective view of a membrane of a reagent strip of the present invention.

Figure 2 is a top view of a sample surface of a direct-reading reagent strip of the present invention.

3 is a plan view of the test surface of the strip of FIG.

Figure 4 is a perspective view of a dual-layered reagent strip of the present invention.

Claims (10)

(a) an anisotropic film having a sample surface having a relatively large gap and a test surface having a relatively small gap and having a sample accommodated on the sample surface and adjusted to pass toward the test surface, and (b) a process for producing (i) glucose oxidase which produces hydrogen peroxide from glucose and oxygen,    (ii) 3-methyl-2-benzothiazolinone hydrazone hydrochloride (MBTH) combined with 3-dimethylaminobenzoic acid (DMAB), which changes color by reacting with hydrogen peroxide; MBTH combined with 3,5-dichloro-2-hydroxybenzene-sulfonic acid (DCHBS); (MBTHSB) combined with [3-methyl-2-benzothiazolinone hydrazone] N-sulfonimine benzenesulfonate monosodium (MBTHSB) combined with ammonium 8-anilino-1-naphthalenesulfonate An indicator comprising a composition and a peroxidase; and  (iii) an acrylic acid polymer having a molecular weight range of 750,000 to 4,000,000 and a concentration range of from 0.1% to 2% w / v, which reduces the influence of red blood cells on the measurement of glucose concentration. doing, A reagent strip for measuring glucose concentration in a whole blood sample containing red blood cells. The reagent strip of claim 1, further comprising a thermoplastic support for the membrane. The reagent strip of claim 1, wherein the indicator comprises peroxidase and MBTHSB-ANS. The reagent strip of claim 1, wherein the molecular weight of the acrylic acid polymer is about 750,000. The reagent strip of claim 1, further comprising a porous carrier layer attached to a sample side of the membrane for receiving the sample and passing a detectable amount thereof to the sample side of the membrane. The reagent strip of claim 1, wherein the reagent further comprises an inhibitor to inhibit color change of the indicator. (a) a method of preparing a microcapsule comprising (i) a sample surface having a relatively large pore and a test surface having a relatively small pore size, (ii) glucose oxidase, peroxidase, Providing a dyestuff or dye couple, and an anisotropic membrane impregnated with a test reagent comprising an acrylic acid polymer having a molecular weight range from 750,000 to 4,000,000 and a concentration range of from 0.1% to 2% w / v, (b) applying blood to the sample side of the membrane and (c) measuring the blood glucose concentration by measuring the color change of the test surface. A method for measuring glucose concentration in a whole blood sample comprising red blood cells. 8. The method of claim 7, wherein the blood glucose concentration range is from 50 to 400 mg / dL. 8. The method of claim 7, wherein the blood hematocrit concentration ranges from 25 to 60%. The reagent strip of claim 1, wherein the concentration of acrylic acid polymer is 0.5% w / v.